B. G. Steinetz

Radioimmunoassay (RIA) of Relaxin in Sera of Various Species Using an Antiserum to Porcine Relaxin

Summary We have investigated the application of a RIA for porcine relaxin to the assay of relaxinlike substances in the blood of various other mammalian species. The cross-reactivity between antiporcine relaxin antibody and the relaxinlike substances in the blood of other mammals during pregnancy was sufficiently high to permit the assay of 0.1–0.5 ml of serum or plasma samples. Nonspecific reactivity was controlled by adding similar volumes of serum or plasma obtained from ovariectomized female or intact male subjects to RIA tubes containing known porcine standards. RIA relaxin levels rose markedly during the last third of pregnancy in rats, mice, and guinea pigs, RIA relaxin was also found in late pregnancy in dog, rhesus and java monkeys, and human beings. The apparent blood levels of hormone found in each species will depend upon their degree of cross-reactivity with the antiporcine relaxin antibody as well as upon their actual concentration. Thus, absolute blood level values should not be taken literally. However, the fluctuations in RIA relaxin observed during the course of gestation in any given species would appear to reflect accurately the relative blood concentrations of the hormone. We thank the following for their invaluable help during the course of this work: Mr. Wilbur Sawyer and Mrs. Margaret Butler for skillful help in various phases of the work; Dr. R. P. Blye, Center for Population Research, NIH, for blood samples obtained from a relaxin-treated baboon; Dr. Wendell H. Niemann, New York University Medical School (LEMSIP), for blood samples obtained from pregnant baboons, java and rhesus monkeys; Dr. Christian Schwabe, Medical University of South Carolina, and Dr. Albert Segaloff, Alton Ochsner Medical Foundation, for human pregnancy serum samples.

Primary structure of the B-chain of porcine relaxin

The sequence of the B-chain of relaxin, and ovarian peptide hormone isolated from ovaries of pregnant sows, has been shown to have the following primary structure: PCA-Ser-Thr-Asn-Asp-Phe-Ile-Lys-Ala-Cys-Gly-Arg-Glu-Leu-Val-Arg-Leu-Trp-Val-Glu-Ile-Cys-Gly-Val-Trp-Ser (2820 daltons). The heterogeneity of relaxin observed during purification procedures is likely to be due to variations in the C-terminal region of the B-chain, in particular the substitution of Gln for Glu20, and the possible addition of arginine or serylarginine at the C terminus. The B-chain exhibited a distribution of sulfhydryl residues relative to one another that is identical to that found in the B-chain of insulin. A similar analogy has already been demonstrated for the A-chains of relaxin and insulin.

SYNERGISTIC EFFECT OF HUMAN RELAXIN AND PROGESTERONE ON HUMAN MYOMETRIAL CONTRACTIONS

In an in vitro human myometrial strip system, both relaxin and progesterone can independently decrease the amplitude of spontaneous myometrial contractions. However, progesterone and relaxin synergize in this action. Doses of relaxin and progesterone which independently are ineffective, together inhibit myometrial contraction amplitude. Relaxin and progesterone are both products of the corpus luteum, a structure necessary for early pregnancy maintenance. The synergistic action of relaxin and progesterone in vitro suggests a similar in vivo physiologic effect in establishing uterine quiescence.

The absolute configuration of the enantiomers of glutethimide and aminoglutethimide

Aminoglutethimide (Elipten® CIBA) was resolved into the optical antipodes I and II. The endocrinological properties and the absolute configuration of both enantiomers I and II were determined. Most of the steroidal synthesis inhibition was found in the (+) enantiomer II. On the basis of circular dichroism, theR-configuration was assigned to the (+) enantiomer II.

Pathway of Absorption of Orally Administered Ethynylestradiol-3-Cy-clopentyl Ether in the Rat as Influenced by Vehicle of Administration.

Summary The pathway of transport of labelled ethynylestradiol (EE) or its 3-cyclo-pentyl ether (EECPE) has been studied following oral administration to rats. The data on lymphatic transport of EE, whether administered as aqueous suspension or dissolved in a lipid vehicle, agree well with previous findings on other steroidal compounds which similarly indicate that: a) the portal system is the major, if not the exclusive pathway of transport of many steroids and b) the pathway of transport is independent of the vehicle of administration. By contrast, the lymphatic transport of EECPE could be significantly increased by use of a lipid vehicle indicating that not only the chemical nature of the compound but also the nature of the vehicle plays an important role in determining pathway of transport.

Studies on Biliary Metabolites of Orally Administered Ethynylestradiol (EE) and its 3-Cyclopentyl Ether (EECPE-Quinestrol)

Summary Large amounts of radioactivity appeared in the bile at 21/2 and 5 hours after a single oral dose of radiolabeled EECPE or EE. Conjugated metabolites (glucuronides and sulfates) predominated in bile although their pattern of distribution differed markedly from that observed in plasma of the same rats. Intestinal washings obtained from treated rats with bile duct cannulas suggested that some alteration of both EECPE and EE may occur in the gut. Thin layer chromatograms of extracts of bile obtained from EE- or EECPE-treated rats did not reveal the presence of unaltered EE or EECPE. However, several of the TLC fractions exerted uterotrophic activity when administered orally to immature rats. The dose response curves were similar to those of EE, suggesting loss of lipophilic properties in the case of EECPE metabolites. None of the fractions were as potent as EE or EECPE. When bile from EECPE-treated donor rats was infused into the small intestine of recipient rats, radioactivity appeared in the plasma and bile of the recipients providing direct evidence of active enterohepatic circulation of metabolites. Little or no radioactivity accumulated in body fat confirming the loss of lipophilic properties following biliary cycling.

Biological and Chromatographic Evidence for the Storage of Ethynylestradiol-3-Cyclopentyl Ether in Rat Brain

Summary The possible storage of estrogenic substances in brains of rats treated with ethynylestradiol (EE) or its 3-cyclopentyl ether (EECPE) was investigated. Twenty-four or 72 hours after a single oral administration of 2 mg of the test steroids, brains were removed and extracted with methylene chloride. The extracts were bio-assayed for uterotrophic potency or subjected to thin layer chromatography. Uterotrophic activity, corresponding to 2.3 and 0.5 μg of the standard, was detected 24 and 72 hours after administration of EECPE. This material corresponded to authentic EECPE in two TLC systems. Similar chromatographic analyses showed that the estrogenic substance, previously detected biologically in body fat of rats treated with EECPE(1), likewise corresponded to the unaltered ether. Neither bioassay nor TLC detected any estrogenic substances in brains or fat of rats treated with EE. The evidence supports the view that EECPE, but not EE, is stored unaltered in brain and body fat and that such storage may account for the enhanced anti-gonadotrophic and uterotrophic potency of the etherified form of the steroid.

Biological Profile of Quingestanol Acetate

Summary Like other 3-enol ethers of δ4δ3-ketosteroids, 17α-ethynyl-19-nor-testo-sterone acetate-3-cyclopentyl enol ether (EN TACP) possesses unusual biological properties. ENTACP is approximately twice as active as its parent ketone (ENTA) in producing endometrial proliferation in the immature estrogen-primed rabbit. In stimulating uterine growth ENTACP behaves like a true estrogen whereas ENTA or 17α-ethynyl-19-nor-testosterone (ENT) act as impeded estrogens, androgens, or progestogens, yielding a shallow dose-response curve. While ENTACP was significantly more androgenic than ENTA or ENT, the three compounds were equipotent in producing masculinization of female fetuses. Although adrenal hypertrophy was noted, neither ENT or ENTACP affected the corticoidogenic response of the adrenals to ACTH. However, when steroid output was corrected for differences in adrenal weight, both compounds either reduced the capacity of the adrenal cortical tissue to respond to ACTH or the increase in adrenal size was not due to an increase in corticosteroid producing tissue. When administered postcoitally, ENTACP was slightly more effective than ENTA in interfering with pregnancy. This may have been related to the inherent estrogenicity of each compound. Unlike methyltestosterone-3-cyclopentyl enol ether, orally administered ENTACP is not stored in body fat and does not have prolonged biological activity.

Studies on the Transport of Estrogens by the Rat Small Intestine in Vivo

Summary Rats intraduodenally administered 17β-estradiol-6,7 -3H (E), ethynylestradiol-6, 7 -3H (EE) or ethynylestradiol-6, 7 -3H-3-cyclopentyl-1-14C ether (EECPE) were killed at 7.5-, 30- or 120-min intervals. Intestinal lumen contents, intestinal wall homogenates and portal plasma were analyzed for free and conjugated steroids. After 7.5 min, more than 80% of the administered dose of E or EE had been absorbed. Characterization of the steroids present in the various ether extractable fractions (free steroid) indicated that, unlike EE, E had undergone rapid metabolic transformation with estrone accounting for 70% of the total radioactivity in the intestinal wall and portal blood. This transformation to the less potent estrone could be responsible for the reduced biological activity of orally administered E.

Effect of aminoglutethimide on serum progesterone and estrogen in the pregnant baboon (Papio SP).

Summary AG was administered orally twice daily for 1 or 3 days to six baboons whose duration of pregnancy ranged from 31 to 99 days. Serum progesterone levels were reduced to as little as 3.2% of the initial concentration in one animal and to 20 % or less in four of the five remaining baboons. Serum estrogen levels were depressed by 50 % or more in three of the four animals in which they were measured. However, pregnancy ensued for at least 3 wk after treatment in all animals. The data suggest that peripheral blood levels of sex steroids may not reflect the critical concentration of hormones required at the uteroplacental juncture for successful pregnancy maintenance. The authors thank Professor Hilton Salhanick of Harvard Medical School for his continuing interest and helpful discussions during the course of this work.